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Bioresource Technology 99 (2008) 32673278

Anaerobic digestion of grass silage in batch leach bed processes


for methane production
A. Lehtomaki

*,1,

S. Huttunen, T.M. Lehtinen, J.A. Rintala

University of Jyvaskyla, Department of Biological and Environmental Science, P.O. Box 35, FI-40014 Jyvaskyla, Finland
Received 6 April 2007; received in revised form 6 April 2007; accepted 6 April 2007
Available online 15 August 2007

Abstract
Anaerobic digestion of grass silage in batch leach bed reactors, with and without a second stage upow anaerobic sludge blanket
(UASB) reactor, was evaluated. Sixty six percent of the methane potential in grass was obtained within the 55 days solids retention time
in the leach bedUASB process without pH adjustment, whereas in the one-stage leach bed process 20% of the methane potential in grass
was extracted. In two-stage operation, adjustment of the pH of inuent to the leach bed reactor to 6 with HCl led to inhibition of both
hydrolysis/acidogenesis and methanogenesis. In the leach bedUASB process 39% of the carbohydrates and 58% of the acid soluble lignin were solubilised within the 49 days of operation, whereas Klason lignin was most recalcitrant. The methane potential of the digestates
varied from 0.141 to 0.204 m3 CH4 kg 1 added volatile solids.
 2007 Elsevier Ltd. All rights reserved.
Keywords: Anaerobic digestion; Energy crop; Methane production; Leach bed; UASB

1. Introduction
Methane-rich biogas produced through anaerobic digestion of organic materials provides a clean and versatile carrier of renewable energy, as methane can be used in
replacement for fossil fuels in both heat and power generation and as a vehicle fuel. Methane production through
anaerobic digestion has been evaluated as one of the most
energy-ecient and environmentally benign ways of producing vehicle biofuel (LBS, 2002). The European Union
(EU) has set a target of increasing the share of biofuels
in vehicles to 5.75% by year 2010 in each member state
(European Parliament, 2003). The utilisation of energy
crops and crop residues for methane production is an interesting option for increasing domestic biofuel production, as
it has been estimated that within the agricultural sector in
*

Corresponding author. Tel.: +358 14 4451 160; fax: +358 14 4451 199.
E-mail address: anni.lehtomaki@pp.inet. (A. Lehtomaki).
1
Presently at Jyvaskyla Innovation Ltd., P.O. Box 27, FI-40101
Jyvaskyla, Finland.
0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.04.072

the EU, 1500 million tons of biomass could be anaerobically digested each year, half of this potential accounted
for by energy crops (Amon et al., 2001). In Finland, for
example, the Ministry of Agriculture has estimated that
by 2012 up to 500 000 hectares (ha), an area corresponding
to about one fourth of all arable land in Finland, could be
dedicated to energy crop production (Vainio-Mattila et al.,
2005).
Energy crops and crop residues can be digested either
alone or in co-digestion with other materials, employing
either wet or dry processes. Energy crops typically have a
high total solids (TS) content of 1050%, and in order to
treat this kind of material in wet processes, the solids must
usually be homogenised and diluted with other materials
high in water content. In the agricultural sector, the most
widely applied solution is to co-digest crop biomass with
animal manures in wet processes (Lehtomaki et al., in
press). Dilution increases the volume to be treated and thus
the energy required for heating and pumping (Ghosh et al.,
2000). Furthermore, oating of the crop materials along
with crust or scum formation has been reported in

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A. Lehtomaki et al. / Bioresource Technology 99 (2008) 32673278

digestion of crop materials in wet processes (Nordberg and


Edstrom, 1997). Moreover, suitable materials for co-digestion may not always be available, and water would then
have to be used for dilution.
The gas production per digester volume (volumetric gas
production) can potentially be increased by operating the
digesters at a higher solids concentration. Batch high solids
reactors, characterised by lower investment costs than those
of continuously fed processes, but with comparable operational costs, are currently applied in the agricultural sector
to a limited extent (Kottner, 2002; Weiland, 2003). In these
systems, digesters are lled with fresh substrate, with or
without addition of inoculum, and allowed to go through
all the degradation steps sequentially. Batch reactors are
often leach bed processes where solids are hydrolysed by circulating leachate over a bed of organic matter. Recirculation of leachate stimulates the overall degradation owing
to more ecient dispersion of inoculum, nutrients and degradation products (Chanakya et al., 1993; Lissens et al.,
2001). Digestion of plant biomass in one-stage leach bed
processes has been seldom reported in the literature (Table
1), but in batch leach bed processes digesting barley straw,
reductions in volatile solids (VS) of 4560% and methane
yields of 0.1590.226 m3 CH4 kg 1 VSadded were obtained
(Torres-Castillo et al., 1995), and in one-stage leach bed
processes fed on a weekly basis with various lignocellulosic
substrates (such as water hyacinth, straw, bagasse, cane
trash etc.) and vegetable wastes, VS removals and biogas
yields ranging from 37% to 78% and from 0.26 to
0.95 m3 biogas kg 1 VSadded, respectively, were reported
(Chanakya et al., 1993, 1997; Ramasamy and Abbasi,
2000) (Table 1).
Batch leach bed processes can also be operated in conjunction with a second stage methanogenic reactor, with
the leachate generated in the rst stage pumped to the
methanogenic reactor for further degradation (Ghosh,
1984). Since the leachate has a low solids content, highrate reactors such as upow anaerobic sludge blanket

reactors (UASBs) or anaerobic lters can be used in the


second stage, and a high solid retention time is achieved
in these reactors through the formation of granules or
attachment of biomass to carriers (Henze and Harremoes,
1983; Lettinga, 1995). Digestion of plant material in processes of this kind has been reported (Table 2), but experiments on digestion of energy crops in these processes are
few. Methane yields and VS removals of 0.27 to
0.39 m3 CH4 kg 1 VSadded and 5960%, respectively, were
obtained in two-stage anaerobic digestion of grass silage
in batch leach bed processes connected to anaerobic lters, in both laboratory (Cirne et al., in press) and pilot
trials (Lehtomaki and Bjornsson, 2006) (Table 2).
In practice, not all of the methane potential in substrates
can be extracted in anaerobic digestion within the reactor
residence time, and if the digestates are stored in uncovered
storage tanks without gas collection, part of this methane
can be lost to the atmosphere through spontaneous degradation, contributing to climate change. Post-methanation
of digestates in covered storage tanks oers the possibility
of both minimizing the potential methane emissions, as well
as contributing to an increase in the methane yields (Kaparaju and Rintala, 2003), as up to 15% more biogas has
been be obtained in post-methanation of digestates from
liquid phase low solids digesters (Weiland, 2003). However,
to our knowledge the methane potential of digestates from
leach bed processes has not been determined previously.
The aim of this study was to evaluate the suitability of
leach bed reactors for methane production from grass
silage. Operation of a one-stage process consisting of a
batch leach bed reactor and a two-stage process with leach
bed reactor in connection with an UASB were compared.
Furthermore, the eect of adjusting the pH of inuent in
the rst stage of the two-stage process was evaluated, and
the extent of degradation of dierent fractions of VS in various stages of digestion was evaluated both by chemical
characterisation and by determining the methane potential
of the digestates.

Table 1
Examples of anaerobic digestion of plant material in one-stage leach bed processes, as reported in the literature
Feedstock

Mode of
feeding

Reactor
volume (l)

T
(C)

Feed TS
(% ww)

VS removal
(%)

Gas production (m3 CH4 kg


m3 biogas kg 1 VSadded)

Barley straw
Water
hyacinth
Paddy straw
Bagasse
Cane trash
Synedrella
Parthenium
Vegetable
waste

Batch
Weekly

220
2

35
2127

3536a
9.4

4560
n.r.

0.159.226b
0.348c

1
2

Weekly
Weekly
Weekly
Weekly
Weekly
Weekly

6
6
6
6
6
11

26
26
26
26
26
35

n.r.
n.r.
n.r.
n.r.
n.r.
n.r.

56.5
37.1
49.8
68.1
78.1
n.r.

0.48a,c
0.83a,c
0.26a,c
0.95a,c
0.71a,c
0.5130.869b

3
3
3
3
3
4

n.r. = not reported.


1: Torres-Castillo et al. (1995), 2: Chanakya et al. (1993), 3: Chanakya et al. (1997), 4: Ramasamy and Abbasi (2000).
a
Values calculated from the data reported.
b
m3 CH4 kg 1 VSadded.
c
m3 biogas kg 1 VSadded.

VSadded or

References

A. Lehtomaki et al. / Bioresource Technology 99 (2008) 32673278

3269

Table 2
Examples of anaerobic digestion of plant material in two-stage processes consisting of a leach bed reactor and a methanogenic reactor, as reported in the
literature
Feedstock

Mode of feeding
in rst stage

Type of reactor as
second stage

Reactor volume rst


stage/second stage (l)

T (C)

Fruit and
vegetable
waste
Fruit and
vegetable
waste
Fruit and
vegetable
waste
Fruit and
vegetable
waste
Fruit and
vegetable
waste
Fruit and
vegetable
waste
Fruit and
vegetable
waste
Fruit and
vegetable
waste
Potato waste
Potato waste
Sugar beet leaves
Unpeeled
potatoes
Peeled potatoes
Sugar beet leaves,
potatoes 1:2
Sugar beet leaves,
potatoes 1:3
Grass waste
Grass silage
Sugar beet
Willow
Grass silage
Sugar beet
Rice straw
Rice straw
Rice straw
Water hyacinth

Batch

UASB-AF

1.3/0.5

35

Batch

UASB-AF

1.3/0.5

Batch

UASB-AF

Batch

Feed TS
(% ww)

VS
removal
(%)

Spec. CH4 yield


(m3 CH4 kg 1 VSadded)

Refs.

83

0.345

35

82

0.355

1.3/0.5

35

87

0.368

UASB-AF

1.3/0.5

35

90

0.383

Daily

UASB-AF

1.3/0.5

35

6.4

72

0.405a

Daily

UASB-AF

1.3/0.5

35

6.4

53

0.294a

Daily

UASB-AF

1.3/0.5

35

6.4

38

0.187a

Daily

UASB-AF

1.3/0.5

35

6.4

27

0.098a

Batch
Batch
Batch
Batch

UASB
AF
AF
AF

2.0/0.84
2.0/1.0
7.6/2.6
7.6/2.6

37
37
3537
3537

19
19
n.r.
n.r.

n.r.
n.r.
n.r.
n.r.

0.39
0.39
0.216a
0.258a

3
3
4
4

Batch
Batch

AF
AF

7.6/2.6
7.6/2.6

3537
3537

n.r.
n.r.

n.r.
n.r.

0.351a
0.402a

4
4

Batch

AF

7.6/2.6

3537

n.r.

n.r.

0.402a

Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Weekly

AF
AF
AF
AF
AF
AF
ASBR
ASBR
ASBR
AF

8000/190
7.6/2.6
7.6/2.6
7.6/2.6
0.75/0.9
0.75/0.9
4.0/4.0
4.0/4.0
4.0/4.0
2.0/0.5

Ambient
37
37
37
37
37
35
35
35
n.r.

92
31.8
20.2
49.5
27
24
92
92
92
9.6

67
59
96
46
60
89
44
45
48
n.r.

0.165a
0.39
0.38
0.16
0.27
0.44
0.19a
0.19a
0.21a
0.181b

5
6
6
6
7
7
8
8
8
9

UASB = upow anaerobic sludge blanket reactor, AF = anaerobic lter, ASBR = anaerobic sequencing batch reactor.
n.r. = not reported. References: 1: Martinez-Viturtia and Mata-Alvarez (1989), 2: Martinez-Viturtia et al. (1995), 3: Parawira et al. (2005), 4: Parawira
et al. (submitted for publication), 5: Yu et al. (2002), 6: Lehtomaki and Bjornsson (2006), 7: Cirne et al. (in press), 8: Zhang and Zhang (1999), 9: Chanakya
et al. (1992).
a
Values calculated from the data reported.
b
m3 biogas kg 1 TSadded.

2. Methods
2.1. Origin of materials
Grass silage was obtained from a farm in central Finland (Kalmari farm, Laukaa) (Table 3). It was prepared
at the farm from grass (75% timothy Phleum pratense,
25% meadow fescue Festuca pratensis) harvested at early

owering stage, chopped with an agricultural precision


chopper after 24 h of pre-wilting and ensiled in a bunker
silo with the addition of a commercial silage additive (lactic
acid bacteria inoculant AIV Bioprot, containing 60% Lactobacillus rhamsonus and 40% Propionibacterium freudenreichii spp. shermanii (Kemira Growhow Ltd.) with a
total count of 5.8 1011 colony-forming units (CFU) g 1,
diluted to 0.7 g l 1 in tap water and applied to the plant

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A. Lehtomaki et al. / Bioresource Technology 99 (2008) 32673278

(Table 3). The UASB was inoculated with granular sludge


obtained from an internal circulation (IC) reactor treating
wastewater from sugar beet and vegetable processing
(Sakyla, Finland).

Table 3
Characteristics of grass silage and inoculum
Parameter

Unit

Grass silage

Inoculum

pH
TS
VS
SCOD
Ntot
NH4-N
Klason lignin
LigninAS
Carbohydrates
Extractives
Proteins
Higher heat content

% ww
% ww
mg g 1 TS
mg g 1 TS
mg g 1 TS
% TS
% TS
% TS
% TS
% TS
MJ kg 1 TS

4.1
25.9
24.0
228
16.9
1.4
13.0
4.1
45.0
8.4
10.4
19.8

7.7
6.6
5.0
189
48.9
17.2
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.

2.2. Experimental set-up


In this study, a one-stage leach bed reactor (LB1) (run 1)
and two two-stage processes combining LB (LB2 and LB3)
and UASB reactors (UASB2, UASB3) (runs 2 and 3) were
used. Furthermore, in order to characterise the changes in
LB material, six LBs in conjunction with a common UASB
(UASB4) were operated (run 4) (Fig. 1).
All the LBs (plastic columns) and UASBs (glass columns) had a liquid volume of 1000 ml and they were operated at 35 (1) C. Leachate from LBs was collected at the
bottom of the reactors in a liquid reservoir (R1) and either
circulated to an UASB or recycled back to the top of the
reactor when internal recirculation was applied. The UASB
euent was collected in another reservoir (R2), from which
it was recirculated to LB (Fig. 1). The biogas produced was
collected from the top of the reactors and the liquid reservoirs into aluminium gas bags. Before starting the present
experiments the UASBs had been inoculated with granular
sludge (see Section 2.1) and operated for two months with

n.d. = not determined.

material in a ratio of 0.5% volume/weight, v/w). In the laboratory, the material was further chopped with a garden
chopper (Wolf Garten SD 180E) to a particle size of
approximately 3 cm and then immediately frozen and
stored at 20 C. Before analysis and feeding to the reactors, the samples were allowed to thaw overnight at 4 C.
The inoculum used to inoculate the one-stage leach bed
reactors and methane potential assays was from a mesophilic farm digester (Laukaa, Finland) treating cow manure and industrial confectionary by-products as substrate

R2
pH adj.

R2

LB1

LB2

R1
pH adj.

R1

LB3

R1

Run 1

Run 3

Run 2
R2

LB

LB

LB

LB

LB

LB

R1

Run 4
Fig. 1. Reactor set-ups in runs 14 (U = UASB, pH adj. = pH adjustment). Dashed lines represent the ow of process liquid during internal recirculation.

A. Lehtomaki et al. / Bioresource Technology 99 (2008) 32673278

articial wastewater at an organic loading rate (OLR) of


5 kg chemical oxygen demand (COD) m 3 d 1.
The LBs (LB1LB3) were lled with 50 g VS (208 g wet
weight, ww) of grass silage mixed with 3.2 g VS (64 g ww)
of inoculum (LB1) or without inoculum (LB2, LB3), after
which 750 ml of tap water was added to ll the reactor (an
initial liquid/solid (L/S) ratio of 17). After each sampling of
leachate an equivalent amount of tap water was added to
the liquid reservoir. When internal recirculation was
applied, the recirculation rate of the leachate was
750 ml d 1. In two-stage operation the OLR to the UASB
was maintained at 5 kg COD m 3 d 1, which determined
the ow rate to both UASB and LBs. The pH adjustments
of the inuents to LBs in runs 1 (pH 7) and 3 (pH 6) were
performed automatically with 1 M NaOH and 1 M HCl.
In run 4 six parallel LBs were lled with 50 g VS (208 g
wet weight) of grass silage without inoculum, after which
250 ml of tap water was added per reactor (1500 ml in
total) (initial L/S ratio 8). The leachate from all six LBs
was collected in a common reservoir (R1) and circulated
from there to the common UASB at 5 kg COD m 3 d 1.
The euent from the UASB was collected in a reservoir
(R2) and circulated back to the top of the LBs so that each
LB received the same liquid at the same ow rate. The LBs
were terminated sequentially during the run to characterise
the residual materials.
The methane potentials of grass silage and digestates
were determined in triplicate batch experiments in 2 l glass
bottles (liquid volume 1.5 l) and in 118 ml glass bottles,
respectively, at 35 1 C. In assays with silage, inoculum
(500 ml) and substrate in a VSsubstrate/VSinoculum-ratio of
1 were added into the bottles, distilled water was added
to achieve a liquid volume of 1.5 l, and NaHCO3 (3 g l 1)
was added as buer. In assays with digestates, the digestates (1 g VS) and inoculum corresponding to 1 g VS were
added into the bottles. The contents of the bottles were
ushed with N2/CO2-gas (70%:30%, Aga Ltd.) for 5 min
and the bottles were then sealed with butyl rubber stoppers.
Bottles were manually mixed before each gas measurement.
Assays with inoculum only were incubated to subtract the
methane yield of the inoculum from those of substrates.
The assays were terminated when CH4 production became
negligible after 94100 days.
The methane potentials of digestates were expressed as
m3 CH4 kg 1 VSadded and m3 CH4 kg 1 VSoriginal. The latter was calculated per VS of substrate originally added to
the reactor, taking into account the VS removal during
reactor operation.
2.3. Analyses and calculations
Methane and volatile fatty acids (VFAs) were measured
by gas chromatograph (GC) (methane: Perkin Elmer Clarus 500 GC with thermal conductivity detector and Supelco
Carboxen 1010 PLOT fused silica capillary column,
30 m 0.53 mm, and VFA: PE Autosystem XL GC with
ame-ionisation detector and PE FFAP column,
TM

3271

30 m 0.32 mm). Operating conditions were for methane:


oven 200 C, injection port 225 C, detector 230 C, and
for VFA: injection port and detector 225 C, oven 100
160 C (20 C/min). Argon (methane) and helium (VFA)
were used as carrier gases.
Metrohm 774 pH-meter was used in all pH measurements. TS and VS were determined according to the Standard Methods (APHA, 1998) and COD according to the
SFS 5504 (Finnish Standards Association, 1988). Total
(Ntot), ammonium nitrogen (NH4-N) and proteins were
determined according to the Tecator application note (Perstorp Analytical/Tecator AB, 1995) with a Kjeltec system
1002 distilling unit (Tecator AB), protein content calculated as 6.25 Ntot. NH4-N and soluble COD (SCOD)
from crop samples were analysed after extraction according to SFS-EN 12457-4 (Finnish Standards Association,
2002) and the samples for NH4-N and SCOD determination were ltered with GF50 glass bre lter papers (Schleicher and Schuell). Extractives were determined by acetone
extraction according to the TAPPI Test Method T 280 pm99 (TAPPI, 2000). For lignin and carbohydrate analyses,
the acetone-extracted samples were hydrolysed according
to the TAPPI Test Method T 249 cm-00 (TAPPI, 2000).
Klason lignin content was measured according to the
TAPPI Test Method T 222 om-98 (TAPPI, 2000). Acid soluble lignin (ligninAS) in hydrolysis ltrate was quantied
spectroscopically (Beckman DU640 Spectrofotometer) on
the basis of ultra-violet absorption at 205 nm using an
absorptivity value of 110 l g 1 cm 1, and total lignin (lignintot) content was calculated as the sum of Klason lignin
and ligninAS. The monosaccharides obtained (arabinose,
galactose, mannose and xylose from the hemicellulose components and glucose from cellulose) were per(trimethylsilyl)ated and analysed with GC (HP 5890 Series II GC
with ame-ionisation detector and a DB-1701 column,
60 m 0.32 mm, Agilent Technologies, J&W Scientic).
Operating conditions were injection port 290 C and detector 300 C. Oven temperature was programmed to begin at
100 C (held for 2 min), rise 2 C/min to 185 C (22 min)
and rise 39 C/min to a nal temperature of 280 C
(15 min). Nitrogen was used as carrier gas. Heat content
was analysed as higher heat content with a bomb calorimeter (IKA-Kalorimeter C400, Janke and Kunkel GmbH).
3. Results
3.1. One-stage leach bed reactors (run 1)
In the one-stage leach bed reactor (LB1) with internal
recirculation and pH adjustment of the inuent (pH 7),
the pH of the LB euent decreased to 4.8 on day 1, but
increased to 6.3 by day 3 and from day 9 onwards varied
between 6.9 and 7.8 for the rest of the 55 days run
(Fig. 2). SCOD in euent reached 15 g l 1 after 1 day, after
which it started to decrease, falling below 2 g l 1 by day 55
(Fig. 2). VFA concentrations peaked at 5.2 g l 1 (total
VFA, TVFA) and accounted to 52% of the SCOD on

A. Lehtomaki et al. / Bioresource Technology 99 (2008) 32673278

Run 1
40

30

30

20

6
5

10

20

40

60

Run 3
9
8
7

40
30
20

6
5
4
3

10
0

pH

SCOD

10

pH
SCOD (gl-1)

SCOD (gl-1)

20

VFA (gl-1)

Run 2

40

20

60

Run 4
40

9
8

30

20

6
5

10

SCOD
pH

4
3

Run 1

40

pH

3272

Run 2
Acetate
Propionate
Butyrate

0
0

20

40

Run 3

60

20

40

60

Run 4

Acetate
2

0
0

20

40

60

Time (d)

Propionate
Butyrate

0
0

20

40

60

Time (d)

Fig. 2. SCOD and VFA concentrations and pH in euent from the leach bed reactors in the one-stage leach bed process (run 1) and in the leach bed
UASB processes, without (runs 2 and 4) and with (run 3) pH adjustment. Dashed lines mark the time when the leach bed reactors were disconnected from
the UASB.

day 13, and decreased steadily from then to <1 g l 1


(Fig. 2).
Methane production and concentration remained low
(less than 5 ml d 1 and 2%, respectively) until day 9, then
started to increase. Methane concentration varied between
34% and 53% from day 16 onwards, while methane production ranged mainly from ca. 30 to 90 ml d 1 (peaking
at 120 ml d 1) and was ca. 30 ml d 1 when the run was
terminated (Fig. 3). During the 55 days run, VS removal
in the LB1 amounted to 34% and the specic methane
yield was 0.060 m3 CH4 kg 1 VSadded and 15 m3 CH4 t 1
ww (tons of wet weight), corresponding to 20% of the
methane potential in grass silage (Table 4). The methane
potential of the LB1 digestate was 0.204 m3 CH4 kg 1 digestate VSadded, and the sum of methane yields from LB1
and from digestate amounted to 71% of the methane
potential in grass (Table 4).

3.2. Two-stage processes: leach bed reactor and UASB


3.2.1. Eect of pH adjustment (runs 2 and 3)
In runs 2 and 3, two leach bed reactors (LB2 and LB3)
were operated initially with internal recirculation and then
in conjunction with UASB reactors. In run 3, the pH of
the inuent to the leach bed reactor (LB3) was adjusted to
6. After 1 day of leachate recirculation, SCOD in both LB
euents had increased to 1112 g l 1, while pH 4.04.2,
and circulation to the UASB was initiated. Circulation
was continued until days 910, when the SCOD in both
LB euents had dropped to below 1 g l 1. After the UASB
was disconnected, the pH in the LB2 euent initially
decreased from 7.3 (day 9) to 6.1, and remained at 6.6 to
6.8 for the rest of the run while in LB3 euent pH varied
between 5.5 and 5.8. Correspondingly, after UASB disconnection SCOD increased in the LB2 euent, peaking at

A. Lehtomaki et al. / Bioresource Technology 99 (2008) 32673278


Leach bed reactor
Run 1

400

3273

UASB
60

300

40

200
20

0
0

20

40
Run 2

400

0
60
60

300
40

0
0

20

40
Run 3

400

0
60

60

300

40

200
20

100
0
0

20

40
Run 4

400

0
60

1200

60

800

40

400

20

10

15

Run 3

0
20

1600

80

1200

60

800

40

400

20

0
0

1600

60

300

80

Gas production (ml d1)

100

CH4 concentration (%)

Gas production (ml d1)

200
20

Run 2

1600

10
Run 4

15

0
20

CH4 concentration (%)

100

80

1200

60

800

40

400

20

40

200
20

100
0
0

20

40

0
60

0
0

Time (d)

10

15

0
20

Time (d)

Fig. 3. Daily gas production and methane concentrations in the one-stage leach bed process (run 1) and in the leach bedUASB processes, without (runs 2
and 4) and with (run 3) pH adjustment. Dashed lines mark the time when the leach bed reactors were disconnected from the UASB. In run 4, values for gas
production in UASB on day 3 are out of scale (3466 ml d 1 CH4 and 2683 ml d 1 CO2, respectively). D CH4 production; CO2 production; h CH4
concentration.

Table 4
Substrate methane potential, specic methane yields, VS removals and digestate methane potentials in the one-stage leach bed process (run 1) and in the
leach bedUASB processes, without (runs 2 and 4) and with (run 3) pH adjustment (average values of replicates standard deviations, where applicable)
Run
Substrate methane potential

m CH4 kg VSadded
m3 CH4 t 1 ww
Specic methane yield
m3 CH4 kg 1 VSadded
m3 CH4 t 1 ww
% of substrate methane potential
VS removal
%
Digestate methane potential
m3 CH4 kg 1 VSadded
m3 CH4 t 1 ww
m3 CH4 kg 1 VSoriginala
Reactor + digestate methane yield of substrate methane potential, %

0.300 0.003
72 1
0.060
15
20
34
0.204 0.013
21 1
0.152 0.010
71

0.300 0.003
72 1
0.197
47
66
55
0.141 0.025
22 4
0.091 0.016
96

0.300 0.003
72 1
0.103
25
34
39
0.160 0.012
19 1
0.115 0.008
73

0.300 0.003
72 1
0.107
26
36
42
n.d.
n.d.
n.d.
n.d.

n.d. = not determined.


a
Calculated per VS of substrate originally added to the reactor, taking into account the VS removal during reactor operation.

3.5 g l 1 on day 23, thereafter decreasing to 1.8 g l 1 at the


end of the run while in the LB3 SCOD remained between
1.5 and 1.8 g l 1 (Fig. 2). The VFAs were mostly higher in
LB2 euent than in LB3 (Fig. 2). The proportion of TVFA

of SCOD was highest, 75%, on day 13 in LB2 euent,


whereas in LB3, the corresponding gure was 42% on day 7.
In both LB2 and LB3 the methane content in the gas
and methane production remained low until days 67

and then started to increase, methane content reaching 47%


in LB2 on day 34% and 43% in LB3 on day 31 (Fig. 3).
Both UASB reactors removed COD initially by more
than 90%, but the removal decreased to 4555% when
the inuent COD dropped to ca. 1 g l 1 (days 910). In
UASB3 the COD reduction uctuated more than in
UASB2. VFAs were not present in the UASB2 euents,
while acetate and propionate were detected in the UASB3
euents in concentrations up to 0.3 g l 1 (data not shown).
Methane concentration in the gas was higher in the UASB2
(6072%) than in UASB3 (4660%). Also methane production was mostly higher in UASB2 than in UASB3, and
continued in both UASBs even after they were disconnected from the LBs (Fig. 3).
VS removal amounted to 55% in LB2 and 39% in LB3
during the 55 and 31 days runs, respectively (Table 4).
The total specic methane yields were 0.197 and
0.103 m3 CH4 kg 1 VSadded in runs 2 and 3, respectively.
Of these methane yields, 80% and 76% in runs 2 and 3,
respectively, originated from the UASB. The methane
potentials of the LB2 and LB3 digestates were 0.141 and
0.160 m3 CH4 kg 1 digestate VSadded, respectively, and
the sum of methane yields in LBs and digestates amounted
to 96% and 73% of the methane potential in grass in runs 2
and 3, respectively (Table 4).

3.2.2. Characterisation of the residues (run 4)


Six LBs installed in parallel and connected to a common
UASB were operated in run 4. The LBs were rst operated
with internal recirculation for 24 h, after which the SCOD
in the LB euent had reached a level of 37 g l 1, and circulation to the UASB was initiated. Circulation to the UASB
was continued until day 17, when the SCOD in the euent
from leach bed reactors had dropped to below 2 g l 1 and
the pH of the LB euent was 7.5 (Fig. 2). After the UASB
was disconnected, the SCOD in the euent from the leach
bed reactors slightly increased, peaking at 3.3 g l 1 on day
20, thereafter varying between 1.5 and 2.6 g l 1 until the
end of the run. VFA concentrations followed a pattern very
similar to that of COD, acetate and propionate peaking at
1.8 and 0.5 g l 1, respectively, on day 3, TVFA corresponding to 2.8 g COD l 1 and decreasing steadily from then on
to <1 g COD l 1 by day 14. After the UASB was disconnected, the pH in the leach bed euents varied between
7.1 and 7.7 for the rest of the run (Fig. 2).
The COD reduction in the UASB was 96% to 99%
until day 10 and then decreased to 47% to 49% as the
SCOD in the leachate declined. Methane concentrations
in the gas produced in UASB varied between 49% and
70%, while the methane concentration in the gas from
the leach bed reactors remained below 1% until day 10,
then started to increase slowly, reaching 14% on day 41
(Fig. 3). The total specic methane yield in run 4 was
0.107 m3 CH4 kg 1 VSadded and 26 m3 CH4 t 1 ww after
49 days of operation, corresponding to 36% of the methane potential in grass silage (Table 4). Of this methane

Reduction (%)

A. Lehtomaki et al. / Bioresource Technology 99 (2008) 32673278


50
40
30

Heat content
reduction

20

VS reduction

10
0

10

20

30

40

50

50

Amount (g)

3274

40

Proteins

30

Extractives
Carbohydrates

20

Acid soluble lignin

10

Klason-lignin

0
0

10

49

Day of sampling
Fig. 4. Reduction in VS and heat content, and amounts of analysed
fractions in untreated grass silage (day 0) and in digestion residues after
dierent periods of digestion in the leach bedUASB process (run 4).

yield, 98% orginated from the UASB, and 2% from leach


bed reactors.
The extent of VS removal was determined each time a
reactor was terminated. After 1 day of leachate recirculation, VS removal had reached 16% (Fig. 4). After day 1,
the reduction in VS slowed down, reaching 30% by the time
methanogenesis had begun in the leach bed reactors (day
17). Total VS removal in run 4 amounted to 42%. The
reduction in heat content correlated well with the VS removals, amounting to 45% at the end of the run (Fig. 4).
The composition of grass was analysed on day 0 and
after 1, 10 and 49 days of digestion. Seventeen percent of
grass TS initially consisted of lignin (Klason lignin and acid
soluble lignin), 45% of carbohydrates, 8% of extractives
and 10% of proteins (Table 3, Fig. 4). After 1 day of digestion, 11% of Klason lignin and 24% of acid soluble lignin
had been removed from the solid residue, whereas proteins,
extractives and carbohydrates had degraded by 34%, 12%
and 10% (Fig. 4). After 10 days of digestion extractives
were the most rapidly removed component, their removal
reaching 59%. At the end of digestion (after 49 days),
74%, 51% and 39% of extractives, proteins and carbohydrates, respectively, had been removed from the solid residue, whereas the removal of Klason lignin and acid soluble
lignin from the solid residue amounted to 17% and 58%,
respectively (Fig. 4). The residue after completion of digestion consisted of 23% (from TS) of lignin, 50% of carbohydrates, 4% of extractives and 9% of proteins.
4. Discussion
Anaerobic digestion of grass silage in leach bed reactors,
with and without a second stage UASB reactor, was eval-

A. Lehtomaki et al. / Bioresource Technology 99 (2008) 32673278

uated, and the highest methane yields were obtained in the


two-stage process without pH adjustment. With this process 66% of the total methane potential in grass silage
was obtained within the 55 days solids retention time,
whereas in the one-stage leach bed process only 20% of
the methane potential in grass silage was extracted during
the corresponding period. In the two-stage process, 76
98% of the total methane yield originated from the UASB,
which clearly shows the advantage of applying a second
stage methanogenic reactor in combination with a leach
bed process.
The methane yields and VS removals obtained in the
present study in the two-stage anaerobic digestion process
employing batch leach bed reactors in the rst stage were of
the same order of magnitude as those reported by Yu et al.
(2002), who obtained a 0.165 m3 CH4 kg 1 VSadded methane yield and 67% VS removal, and Cirne et al. (in press),
who reported a 0.27 m3 CH4 kg 1 VSadded methane yield
and 60% VS removal, in laboratory batch leach bed processes connected to anaerobic lters digesting grass waste
(Yu et al., 2002) and grass silage (Cirne et al., in press,
Table 2). Lehtomaki and Bjornsson (2006) obtained 59%
VS removal and a methane yield of 0.39 m3 CH4 kg 1
VSadded after 50 days of digestion of grass silage in pilot
batch leach bed processes connected to anaerobic lters
(Table 2). The higher methane yields reported in the latter
study compared with those obtained in the present study
were most likely due to dierences in the composition of
the grass mixtures used as substrates, since the grass used
in the present study had lower biodegradability, as indicated by its lower methane potential and higher lignin concentration (Lehtomaki and Bjornsson, 2006). Lignin is
known to be poorly degraded in anaerobic conditions,
and the intense cross-linking of lignin with cellulose and
hemicellulose also limits the degradation of these bre fractions (Fan et al., 1981). Nutrient restriction on microbial
degradation due to the lower nitrogen content of the grass
may also have been a cause for the lower methane yields in
the present study.
The volumetric methane yields in one- and two-stage
leach bed processes were low compared with previously
reported yields in either batch or continuously fed wet processes. We have previously operated wet processes (continuously stirred tank reactors, CSTRs) co-digesting grass
silage, similar to the one used in the present study, with
cow manure with up to 40% of grass in the feed VS, and
obtained up to 53% VS removal and a methane yield of
0.268 m3 CH4 kg 1 VSadded, corresponding to 105% of the
total methane potential in the substrates (Lehtomaki
et al., in press), whereas in the present study, up to 66%
of the total methane potential in grass was obtained in
the leach bedUASB process. The higher methane yields
obtained with co-digestion can be partly explained by synergy eects due to a more balanced nutrient composition in
the feed, but also by microbial adaptation, which is likely
to be enforced by the semi-continuous feeding in CSTRs
(Lehtomaki et al., in press) as opposed to the batch pro-

3275

cesses applied in the present study. Furthermore, in the


two-stage process described in this report, no inoculum
addition was done in the rst stage. Inoculating the batch
reactors with digestate from previous runs would enable
continuous adaptation of microbes to the degradation of
the substrate and would be likely to enhance the extent
of degradation and methane production also in batch
processes.
In the one-stage leach bed process, 83% of the extracted
COD was converted to methane, whereas the corresponding gure for the two-stage operation was 9295%. The
low COD extraction rate in the one-stage operation was
apparently due to the high SCOD and VFA concentrations
in the recirculated leachate (SCOD and TVFA up to 15
and 7 g l 1, respectively), which can cause inhibition of
hydrolysis and acidogenesis (Vavilin et al., 2003), whereas
in the two-stage operation, the UASB eciently removed
SCOD and VFA from the leachate (up to 99% SCOD
reduction), as a result of which the UASB euent returned
to the batch leach bed reactor was low in SCOD and VFA
(mostly <1 g l 1), resulting in turn in more ecient extraction of grass SCOD. VFA accumulation was apparently
the cause of the lower methane yield and lower VS removal
also in run 4 with six parallel leach bed reactors, where the
lower L/S ratio (8) applied resulted in higher SCOD and
VFA concentrations in the leachate as opposed to the corresponding run with a L/S ratio twice as high (17 in run 2).
Grasses are primarily composed of cellulose, hemicelluloses and lignin, the polysaccharides and lignin accounting
together for 62% of the grass TS, as analysed in the present
study. The carbohydrate and lignin content of grass in the
present study was close to that previously reported for boreal timothy-based grasses (carbohydrates 3743% TS, lignin 1619% TS, Viinikainen et al., submitted for
publication). In total, 39% of the carbohydrates were
removed in the leach bedUASB process within the 49 days
of operation. Proteins were the most rapidly hydrolysable
component in grass, as they were degraded to the highest
extent after 1 day of liquid recirculation, whereas extractives were the most solubilised component after 10 and
49 days of operation. The apparent loss of lignin in leach
bed digesters fed with grass silage was most probably due
to solubilisation rather than degradation, as also suggested
by Kivaisi et al. (1990), as lignin is known to be refractory
and poorly degraded in anaerobic conditions (Fan et al.,
1981). However, in the present study it was shown that
more than half of the acid soluble lignin was solubilised
after 49 days of digestion in a leach bed digester fed with
grass silage, whereas Klason lignin was the most recalcitrant component of those determined in the present study.
In the two-stage operation, adjustment of the pH of
inuent to the leach bed reactor to 6 with HCl led to inhibition in both the leach bed reactor and the UASB. Inhibition
of hydrolysis and acidogenesis in the leach bed process were
indicated by the low SCOD values and the low share of
TVFA of SCOD in the leachate, whereas inhibition of methanogesis in the UASB was indicated by the presence of

3276

A. Lehtomaki et al. / Bioresource Technology 99 (2008) 32673278

VFAs in the UASB euent and by the lower and uctuating methane concentration in the gas from the UASB (varying between 46% and 60%) compared with that in the
corresponding run without pH adjustment, despite the similar UASB loading rates in the two experiments. The low VS
removal and the high post-methanation potential of digestate from the run with pH adjustment (run 3) indicated a
much lower extent of degradation than in the corresponding
experiment without pH adjustment (run 2), with the result
that the total specic methane yield from the run with pH
adjustment (run 3) remained much lower than in the corresponding run without pH adjustment (run 2) despite the
similar UASB loading rates. Due to the problems in the
UASB, the run with pH adjustment (run 3) was terminated
after only 31 days of operation. pH values of around 6 have
been reported optimal for the functioning of the extracellular cellulase enzymes produced by hydrolytic bacteria (Sleat
and Mah, 1987), and therefore it was assumed that pH
adjustment to 6 could be advantageous in a leach bed process. However, lowering the pH below neutral did not
clearly enhance the rate of hydrolysis in this and some previous experiments (Veeken et al., 2000; Dinamarca et al.,
2003; Babel et al., 2004). Moreover, chloride ion has been
reported to give rise to toxic eects in anaerobic wastewater
treatment (Mendez et al., 1992; Vijayaraghavan and
Ramanujam, 1999), and thus it is possible that the low
methane yields and VS removal in run 3 were caused by
inhibitory eects due to the application of hydrochloric acid
in pH adjustment. However, Wujcik and Jewell (1980)
found no inhibitory eect due to increased chloride concentrations (added as NaCl) in high solid digesters digesting
newsprint paper and dairy manure, and Zhang et al.
(2005) did not report any inhibitory eects in hydrolysis
and acidogenesis of kitchen waste when hydrochloric acid
was used in pH adjustment.
The inoculation ratio applied in the one-stage leach bed
processes digesting grass silage (6% of inoculum of total
VS) was apparently too low for an ecient extraction of
the methane potential in the substrate, as indicated by
the low specic methane yield and VS removal during reactor operation, as well as by the high post-methanation
potential in the digestate. Torres-Castillo et al. (1995, Table
1) studied digestion of barley straw in batch leach bed reactors with varying inoculum concentrations (212% of VS),
and the highest gas production was obtained in the reactor
where the share of inoculum was highest (12% of VS:
0.226 m3 CH4 kg 1 VSadded). However, the dierence in
gas production between the reactors inoculated with 12%
and 6% of inoculum (0.211 m3 CH4 kg 1 VSadded) was only
minor and overshadowed by the lower volumetric gas production at the higher inoculum application ratios. Therefore, the authors recommended the use of 6% of
inoculum of total VS (Torres-Castillo et al., 1995). In digestion of wheat straw in batch leach bed reactors with varying inoculum concentrations (520% of inoculum of total
VS), the dierence in reactor performance using a large
or small addition of inoculum was insignicant after a

few days of hydrolysis, and an inoculum concentration of


up to 5% was suggested sucient for a proper start-up
(Llabres-Luengo and Mata-Alvarez, 1988). However, grass
silage is a more biodegradable substrate than straw, as indicated by the higher methane potential and the higher
amounts of readily available soluble compounds in grass
compared with straw (Lehtomaki et al., in press). Therefore, the inoculation ratio previously recommended for
the digestion of straw was too low for that of grass silage,
pointing to the need to optimise the substrate/inoculum
ratios for batch processes digesting energy crops.
The present results showed that the digestates still contained degradable material with signicant methane potential, which, if completely recovered, would correspond to
up to 0.204 m3 CH4 kg 1 VSadded of digestate and 3272%
of the total methane production (sum of methane production in reactors and in post-methanation), the proportion
being highest after digestion of grass in the one-stage leach
bed process. If not recovered, part of this post-methanation
potential can be lost as atmospheric methane emissions
due to spontaneous degradation, the extent of which would
be strongly dependant on the ambient temperatures
(Kaparaju and Rintala, 2003). Digestates from CSTRs
co-digesting energy crops and crop residues with cow
manure had post-methanation potentials of 0.133
0.197 m3 CH4 kg 1 VSadded and 38 m3 CH4 t 1 ww of
digestate after 100 days post-methanation at 35 C (Lehtomaki et al., in press). Thus, the post-methanation potentials of digestates from one- and two-stage leach bed
processes were of the same order of magnitude as those
from CSTRs when calculated per VS of digestate. However, owing to the high TS concentrations (1217% in the
present study) of digestates from leach bed processes compared with those from CSTRs (35% according to Lehtomaki et al. (in press)), the values for post-methanation
potential in the present study were of an order of magnitude higher than those obtained in post-methanation of
digestates from CSTRs when calculated per wet weight.
Applying post-methanation enabled long total retention
times (131155 days) in the present experiment, yielding
in total 7196% of the grass methane potential as measured
in the batch methane potential assays with 94 days retention time.
5. Conclusions
Anaerobic digestion of grass silage in leach bed reactors,
with and without a second stage UASB reactor, was evaluated, and the highest methane yields were obtained in the
two-stage process without pH adjustment. With this process
66% of the total methane potential in grass silage was
obtained within the 55 days solids retention time, whereas
in the one-stage leach bed process only 20% of the methane
potential in grass silage was extracted during the corresponding period. In the two-stage process, up to 98% of
the total methane yield originated from the UASB, demonstrating the advantage of applying a second stage methano-

A. Lehtomaki et al. / Bioresource Technology 99 (2008) 32673278

genic reactor in combination with a leach bed process. In


the two-stage operation, adjustment of the pH of inuent
to the leach bed reactor to 6 with HCl inhibited both hydrolysis/acidication and methanogenesis. The leach bed
UASB process removed 39% of the carbohydrates, while
more than half of the acid soluble lignin was solubilised,
whereas Klason lignin was the most recalcitrant component
of those determined in the present study. The digestates
still contained degradable material with signicant
methane potential, which, if completely recovered, would
correspond to 0.1410.204 m3 CH4 kg 1 VSadded and 19 to
22 m3 CH4 t 1 ww of digestate.
Acknowledgements
The authors wish to thank EU Sixth Framework Programme (Project SES6-CT-2004-502824) and the Finnish
Graduate School for Energy Technology for providing
funding for this work, and farmer E. Kalmari for kindly
providing the substrates and Lannen Tehtaat plc for providing the granular sludge. Furthermore, Ms. L. Malkki
and Ms. S. Rissanen are acknowledged for their help in
maintaining the reactors and conducting the laboratory
analyses.
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